DNAs encoding chimeric immunoglobulin light or heavy chains and fragments thereof having variable regions derived from monoclonal antibody 7E3, and vectors and host cells comprising same

ABSTRACT

Platelet-specific, chimeric immunoglobulin and immunoglobulin fragments are described. The chimeric molecules are made up of a nonhuman antigen binding region and a human constant region. Preferred immunoglobulins are specific for glycoprotein IIb/IIIa receptor in its complexed form; they block ligand binding to the receptor and prevent platelet aggregation. The immunoglobulins are useful in anti-thrombotic therapy when administered alone or in conjunction with thrombolytic agents, as well as in thrombus imaging.

RELATED APPLICATIONS

This application is a division of application Ser. No. 08/375,074 filedJan. 17, 1995 which is a File Wrapper Continuation of 07/771,656 filedOct. 4, 1991, which is a Continuation-in-Part of 07/195,720, filed May18, 1988 all abandoned.

BACKGROUND OF THE INVENTION

Platelet aggregation is an essential event in the formation of bloodclots. Under normal circumstances, blood clots serve to prevent theescape of blood cells from the vascular system. However, during certaindisease states, clots can restrict or totally occlude blood flowresulting in cellular necrosis.

For example, platelet aggregation and subsequent thrombosis at the siteof an atherosclerotic plaque is an important causative factor in thegenesis of conditions such as angina, acute myocardial infarction, andreocculusion following successful thrombolysis and angioplasty. Heartattack patients are typically treated with thrombolytic agents such astissue plasminogen activator or streptokinase, which dissolve the fibrincomponent of clots. A major complication associated with fibrinolysis isreocclusion based on platelet aggregation which can result in furtherheart damage. Since glycoprotein (GP)IIb/IIIa receptors are known to beresponsible for platelet aggregation, reagents which block thesereceptors are expected to reduce or prevent reocclusion followingthrombolytic therapy and to accelerate the rate of thrombolysis. Suchreagents are also expected to be useful in therapy of othervaso-occlusive and throboembolic disorders.

One approach to blocking platelet aggregation involves monoclonalantibodies specific for GPIIb/IIIa receptors. A murine monoclonalantibody, designated 7E3, that inhibits platelet aggregation and appearsuseful in the treatment of human thrombotic diseases is described inpublished European Patent Application Nos. 205,207 and 206,532. It isknown in the art that murine antibodies have characteristics which mayseverely limit their use in human therapy. As foreign proteins, murineantibodies may elicit immune reactions that reduce or destroy theirtherapeutic efficacy and/or evoke allergic or hypersensitivity reactionsin patients. The need for readministration of such therapeuticmodalities in thromboembolic disorders increases the likelihood of thesetypes of immune reactions.

Chimeric antibodies consisting of non-human binding regions joined tohuman constant regions have been suggested as a means to circumvent theimmunoreactivity problems of murine antibodies. See Proc. Natl. Acad.Sci. USA, 81:6851 (1984) and PCT Application No. PCT/GB85 00392. Sincethe constant region is largely responsible for immunoreactivity of anantibody molecule, chimeric antibodies with constant regions of humanorigin should be less likely to evoke an anti-murine response in humans.However, it is unpredictable whether the joining of a human constantregion to a murine binding region of a desired specificity will reduceimmunoreactivity and/or alter the binding capability of the resultingchimeric antibody.

SUMMARY OF THE INVENTION

This invention pertains to a platelet-specific chimeric immunoglobulincomprising a variable or antigen binding region of non-human origin anda constant region of human origin. The chimeric immunoglobulins can bespecific for GPIIb/IIIa receptor or other platelet components. Theseantibodies bind to platelets and can block platelet aggregation and thusare useful as antithrombotic agents and to prevent or reduce reocclusionfollowing thrombolysis.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B are schematic representations of the plasmids p7E3V.sub.κhC.sub.κ and p7E3V_(H) hC_(G4), which carry the chimeric gene constructsencoding the light and heavy chains, respectively, of a chimeric 7E3immunoglobulin.

FIG. 2 shows the binding of the chimeric 7E3 immunoglobulin encoded byvectors p7E3V.sub.κ hC.sub.κ and p7E3V_(H) hC_(G4) to platelets.

FIG. 3 shows the inhibition of platelet aggregation by a chimeric 7E3(c7E3) immunoglobulin.

FIG. 4 is a graphic illustration of a plot of the plasma antibodyconcentration (ng/mL) versus time (days) which demonstrates the rapidinitial clearance of c7E3 Fab (γ₁, κ) from the plasma in three patientswith stable coronary disease, following a 0.25-mg/kg dose of c7E3 Fabadministered intravenously as a five minute infusion.

FIGS. 5A-5C are graphs summarizing the effect on platelet activity of asingle bolus dose of chimeric 7E3 Fab (0.15 mg/kg, 0.20 mg/kg or 0.25mg/kg) 2 hours after administration of antibody (γ₁, κ). A dose responseis evident when platelet activity is assayed in terms of receptorblockade (FIG. 5A, top), platelet aggregation (FIG. 5B, middle), andbleeding time (FIG. 5C, bottom). The lines represent median values.

FIGS. 6A-6C are graphs illustrating the duration of anti-platelet effectof chimeric 7E3 Fab (γ₁, κ) administered prior to angioplasty in a bolusdose of 0.25 mg/kg. The lines indicate the median values from time zeroat baseline through 24 hours for receptor blockade (FIG. 6A, top),platelet aggregation (FIG. 6B, middle), and bleeding time (FIG. 6C,bottom).

FIGS. 7A-7C are graphs summarizing the anti-platelet activity of a 0.25mg/kg bolus dose followed by a 12 hour continuous infusion (10μg/minute) of chimeric 7E3 Fab (γ₁, κ) in 11 patients. The linesrepresent median values determined for percent receptor blockade (FIG.7A, top), percent of pre-dose (baseline at time zero) plateletaggregation (FIG. 7B, middle), and bleeding times (FIG. 7C, bottom).

FIG. 8 is an illustration of the absolute change in hematocrit frombaseline to a time 24 hours following the end of infusion for 47patients described in Example 4.

DETAILED DESCRIPTION OF THE INVENTION

The chimeric immunoglobulins of the present invention are comprised ofindividual chimeric heavy and light immunoglobulin chains. The chimericheavy chain is comprised of an antigen-binding region derived from theheavy chain of a non-human antibody specific for platelets (e.g.,specific for the GPIIb/IIIa receptor) linked to a human heavy chainconstant region. The chimeric light chain comprises an antigen bindingregion derived from the light chain of the non-human antibody linked toa human light chain constant region.

The present immunoglobulins can be monovalent, divalent or polyvalent.Monovalent immunoglobulins are dimers (HL) formed of a chimeric heavychain associated through disulfide bridges with a chimeric light chain.Divalent immunoglobulins are tetramers (H₂ L₂) formed of two dimersassociated through at least one disulfide bridge. Polyvalentimmunoglobulins can also be produced, for example, by employing a heavychain constant region that aggregates (e.g., μ heavy chain constantregions). Chimeric immunoglobulin fragments such as Fab, Fab' or F(ab')₂can also be produced.

The non-human antigen binding regions of the chimeric immunoglobulin arederived from immunoglobulins specific for platelets. Preferredimmunoglobulins are specific for platelet GPIIb/IIIa receptors and canblock ligand binding to the glycoprotein IIb/IIIa receptor complex.

Thrombosis begins with the adhesion of platelets at sites of vessel wallinjury. The adhesion of platelets is mediated by platelet surfacereceptors which bind to extracellular matrix proteins in the exposedsubendothelium, such as von Willebrand factor, collagen, fibronectin,vitronectin, and laminin. Platelet adhesion results in a monolayer ofplatelets. Subsequently, platelet activation occurs in response toagonists such as epinephrine, ADP, collagen, and thrombin. Activationleads to the exposure of the glycoprotein IIb/IIIa receptor (GPIIb/IIIa)on the platelet surface. GPIIb/IIIa on activated platelets is thenavailable to bind to fibrinogen, which can mediate platelet aggregation.The binding of GPIIb/IIIa to other adhesive proteins, such as vonWillebrand factor can also cause platelet cross-linking and aggregation.Thus, the binding of adhesive molecules, such as fibrinogen or vonWillebrand factor, to GPIIb/IIIa to cause aggregation of platelets is acommon step in thrombus formation, making the GPIIb/IIIa receptor anattractive target for therapeutic agents which can interfere with theinteraction of GPIIa/IIIb with these molecules. Furthermore, by use ofan anti-GPIIb/IIIa chimeric antibody, the aggregation of activatedplatelets is expected to be inhibited, without interfering with theinitial adhesion of platelets. This selective inhibition of plateletaggregation may be desirable because platelet adhesion, withoutaggregation, may contribute to maintaining hemostasis.

Examples of suitable antibodies specific for platelets include 7E3 and10E5. See European Patent Application Nos. 205,207 and 206,532, theteachings of which are incorporated herein. The 7E3 antibody (orantibody reactive with the same or a functionally equivalent epitope) isespecially preferred because it is specific for the the complexed formof the GPIIb/IIIa receptor. Other antibodies specific for the GPIIb/IIIareceptor (antigen recognized by 7E3), such as those specific for eitherthe IIb or IIIa components, can also be used. Antibodies specific forother platelet antigens can be employed. For example, antibodiesreactive with platelet a granule membrane protein GMP-140 such as S12antibody (J. Biol. Chem. 259:9799-9804 (1984)) can be used.

Preferably, the antigen binding region will be of murine origin becausemurine antibodies against platelets and particularly, GPIIb/IIIareceptors, are available or can be produced in murine systems. Otheranimal or rodent species provide alternative sources of antigen bindingregions.

The constant regions of the chimeric antibodies are derived from humanimmunoglobulins. The heavy chain constant region can be selected fromany of the five isotypes alpha, delta, epsilon, gamma or mu. Further,heavy chains of various subclasses (such as the IgG subclasses of heavychains) are responsible for different effector functions and thus, bychoosing the desired heavy chain constant region, chimeric antibodieswith desired effector function can be produced. Preferred constantregions are gamma 1 (IgG1), gamma 3 (IgG3) and gamma 4 (IgG4). The lightchain constant region can be of the kappa or lambda type.

In general, the chimeric antibodies are produced by preparing, for eachof the light and heavy chain components of the chimeric immunoglobulin,a fused gene comprising a first DNA segment that encodes at least thefunctional portion of the platelet-specific variable region of nonhumanorigin linked (e.g., functionally rearranged variable region withjoining segment) to a second DNA segment encoding at least a part of ahuman constant region. Each fused gene is assembled in or inserted intoan expression vector. Recipient cells capable of expressing the geneproducts are then transfected with the genes. The transfected recipientcells are cultured under conditions that permit expression of theincorporated genes and the expressed immunoglobulins or immunoglobulinchains are recovered.

Genes encoding the variable region of Ig light and heavy chains can beobtained from lymphoid cells that produce the platelet-specificantibodies. For example, the hybridoma cell lines that produce antibodyagainst the GPIIb/IIIa receptor provide a source of immunoglobulinvariable region for the present chimeric antibodies. Other rodent celllines are available. Cell lines can be produced by challenging a rodentwith a human platelet or a GPIIb/IIIa receptor-containing component orfraction of platelet, forming fused hybrid cells betweenantibody-producing cells and a myeloma cell line, cloning the hybrid andselecting clones that produce antibody against platelet or glycoproteinIIb/IIIa receptor.

Constant regions can be obtained from human antibody-producing cells bystandard cloning techniques. Alternatively, because genes representingthe two classes of light chains and the five classes of heavy chainshave been cloned, constant regions of human origin are readily availablefrom these clones. Chimeric antibody binding fragments such as F(ab')₂and Fab fragments can be prepared by designing a chimeric heavy chaingene in truncated form. For example, a chimeric gene encoding a F(ab')₂heavy chain portion would include DNA sequences encoding the CH₁ domainand hinge region of the heavy chain. Alternatively, such fragments canbe obtained by enzymatic cleavage of a chimeric immunoglobulin. Forinstance, papain or pepsin cleavage can generate Fab or F(ab')₂fragments, respectively.

Preferably, the fused genes encoding the light and heavy chimeric chains(or portions thereof) are assembled in two different expression vectorsthat can be used to cotransfect a recipient cell. Each vector containstwo selectable genes--one for selection in a bacterial system and onefor selection in a eukaryotic system--each vector having a differentpair of genes. These vectors allow production and amplification of thefused genes in bacterial systems, and subsequent cotransfection ofeukaryotic cells and selection of the cotransfected cells. Examples ofselectable genes for the bacterial system are the genes that conferampicillin resistance and the gene that confers chloramphenicolresistance. Two selectable genes for selection of eukarytoictransfectants are preferred: (i) the xanthine-guaninephosphoribosyltransferase gene (gpt), and (ii) the phosphotransferasegene from Tn5 (designated neo). Selection with gpt is based on theability of the enzyme encoded by this gene to use xanthine as asubstrate for purine nucleotide synthesis; the analogous endogenousenzyme cannot. In a medium containing xanthine and mycophenolic acid,which blocks the conversion of inosine monophosphate to xanthinemonophosphate, only cells expressing the gpt gene can survive. Theproduct of the neo blocks the inhibition of protein synthesis ineukarytoic cells caused by the antibiotic G418 and other antibiotics ofits class. The two selection procedures can be used simultaneously orsequentially to select for the expression of immunoglobulin chain genesintroduced on two different DNA vectors into a eukaryotic cell.

The preferred recipient cell line is a myeloma cell. Myeloma cells cansynthesize, assemble and secrete immunoglobulins encoded by transfectedIg genes. Further, they possess the mechanism for glycosylation of theimmunoglobulin. A particularly preferred recipient cell is aIg-non-producing myeloma cell line such as Sp2/0. These cell linesproduce only the immunoglobulin encoded by the transfectedimmunoglobulin genes. Myeloma cells can be grown in culture or in theperitoneum of mice where secreted immunoglobulin can be obtained fromascites fluid. Other lymphoid cells such as B lymphocytes or hybridomacells can serve as suitable recipient cells.

Several methods exist for transfecting lymphoid cell with vectorscontaining immunoglobulin encoding genes. A preferred way of introducingDNA into lymphoid cells is by electroporation. In this procedurerecipient cells are subjected to an electric pulse in the presence ofthe DNA to be incorporated. See e.g., Potter et al., Proc. Natl. Acad.Sci. USA 81:7161 (1984). Another way to introduce DNA is by protoplastfusion. In this method, lysozyme is used to strip cell walls frombacteria harboring the recombinant plasmid containing the chimeric Iggene. The resulting spheroplasts are fused with myeloma cells withpolyethylene glycol. After protoplast fusion, the transfectants areselected and isolated. Another technique that can be used to introduceDNA into many cell types is calcium phosphate precipitation.

The chimeric immunoglobulin genes can also be expressed in nonlymphoidcells such as bacteria or yeast. When expressed in bacteria, theimmunoglobulin heavy chains and light chains become part of inclusionbodies. Thus, the chains must be isolated and purified and thenassembled into functional immunoglobulin molecules. Other strategies forexpression in E. coli are available (see e.g., Pluckthun, A.,Bio/Technology 9:545-551 (1991); Skerra, A. et al., Bio/Technology9:273-278 (1991)), including secretion from E. coli as fusion proteinscomprising a signal sequence.

Utility of Platelet-specific Chimeric Immunoglobulin

The chimeric platelet-specific antibodies of this invention are usefulas antithrombotic therapeutic agents. The chimeric antibodies (orfragments thereof) can be used to inhibit platelet aggregation andthrombus formation. The antibodies can also be used to inhibit cyclicflow variations which are caused by platelet aggregation, and which mayprecede thrombus formation or reformation. The antibodies can be used ina variety of situations where thrombus formation or reformation(reocclusion) is to be prevented. For instance, the antibody can beadministered to an individual (e.g., a mammal such as a human) toprevent thrombosis in pulmonary embolism, transient ischemic attacks(TIAs), deep vein thrombosis, coronary bypass surgery, surgery to inserta prosthetic valve or vessel (e.g., in autologous, non-autologous orsynthetic vessel graft). The antibodies of the present invention canalso be administered to an individual to prevent platelet aggregationand thrombosis in angioplasty procedures performed by balloon, coronaryatherectomy, laser angioplasty or other suitable methods. Antibody canbe administered prior to the angioplasty procedure (pre-angioplasty),during angioplasty, or post-angioplasty. Such treatment can preventthrombosis and thereby reduce the rate of thrombotic complicationsfollowing angioplasty, such as death, myocardial infarction, orrecurrent ischemic events necessitating PTCA or coronary bypass surgery.

For instance as shown in Example 4, administration of a chimericanti-platelet antibody (chimeric 7E3 Fab fragment) as adjuvant therapyprior to angioplasty (percutaneous transluminal coronary angioplasty,PTCA) increased bleeding times and reduced agonist-induced plateletaggregation as assayed by ex vivo platelet aggregation assays. Theresults of the experiments reported in Examples 4 and 5 also suggestthat blockade of platelet GPIIb/IIIa and inhibition of aggregation byc7E3 antibody (an Fab fragment) translates into in vivo antithromboticefficacy in humans.

The aggregation of platelets activates the coagulation cascade andproduces a more stable fibrin meshwork and occlusive clot, which can belysed by thrombolytic agents. The antibody can be administered to anindividual (e.g., a human) alone or in conjunction with a thrombolyticagent, such as as a plasminogen activator (e.g., tissue plasminogenactivator, urokinase, or streptokinase, recombinant tissue plasminogenactivator) or an anticoagulant or anti-platelet agent, such as aspirin,heparin, or a coumarin anticoagulant (e.g., warfarin), to prevent orreduce reocclusion that can occur after thrombolysis and to accelerateclot lysis. The antibody or fragment can be administered before, alongwith or subsequent to administration of the thrombolytic agent oranticoagulant, in amounts sufficient to prevent platelet aggregationthat can result in reocclusion.

An effective amount (e.g., an amount sufficient for inhibition ofplatelet aggregation and thereby of inhibition of thrombus formation) ofthe antibody or antibody fragment can be given parenterally, preferablyintravenously, in a pharmaceutically acceptable vehicle such as sterilesaline. Buffered media may be included. The antibody formulation cancontain additional additives, such as a stabilizer (e.g., Polysorbate80, USP/NF). The antibody can be administered in a single dose,continuously, or in multiple infusions (e.g., a bolus injection,followed by continuous infusion). Alternatively, the antibody could beadministered by a controlled release mechanism (e.g., by a polymer orpatch delivery system) or by another suitable method. The amount to beadministered will depend on a variety of factors such as the clinicalsymptoms, weight of the individual, whether other drugs (e.g.,thrombolytic agents) are administered.

During repeat therapy with anti-platelet antibodies, drug-inducedthrombocytopenia may occur; this may be a result of the body recognizingthe antibody-coated platelets as foreign proteins, raising antibodiesagainst them, and then clearing them via the reticuloendothelial systemmore rapidly than normal. Because of the uniquely high density of theGPIIb/IIIa receptor on the platelet surface (˜80,000 receptors perplatelet) and the large number of platelets in the circulation (˜0.25-0.5×10⁶ per μl), thrombocytopenia may be an important complication oftreatment with anti-platelet antibodies. The use of a chimericanti-platelet (e.g., anti-GPIIb/IIIa) antibody can avoid this problem.It is predicted that the majority of the murine component of thechimeric antibody will be bound to the platelet (e.g., via theGPIIb/IIIa receptor) and thus will be inaccessible to the immune system,rendering the chimeric antibody functionally indistinguishable from ahuman antibody directed against the same epitope. Therefore, thechimeric antibody is expected to be non-immunogenic in spite of themurine antigen binding region. In addition, the chimeric anti-plateletantibodies of the present invention may minimize (reduce or prevent) thethrombocytopenia which might otherwise occur on administration of ananti-platelet antibody.

The platelet-specific chimeric immunoglobulins of this invention arealso useful for thrombus imaging. For this purpose, antibody fragmentsare generally preferred. As described above, chimeric heavy chain genecan be designed in truncated form to produce a chimeric immunoglobulinfragment (e.g., Fab, Fab', or F(ab')₂) for immunoscintigraphic imaging.These molecules can be labeled either directly or through a coupledchelating agent such as DTPA, with radioisotopes such as ¹³¹ Iodine, ¹²⁵Iodine, ^(99m) Technetium or ¹¹¹ Indium to produceradioimmunoscintigraphic agents. Alternatively, a radiometal binding(chelating) domain can be engineered into the chimeric antibody site toprovide a site for labeling. Thus, a chimeric immunoglobulin can bedesigned as a protein that has a nonhuman platelet-specific variableregion, a human constant region (preferably truncated), and a metalbinding domain derived from a metal binding protein, such asmetallothionein.

The platelet-specific chimeric immunoglobulin is administered to apatient suspected of having thrombus. After sufficient time to allow thelabeled immunoglobulin to localize at the thrombus site, the signalgenerated by the label is detected by a photoscanning device such as agamma camera. The detected signal is then converted to an image of thethrombus. The image makes it possible to locate the thrombus in vivo andto devise an appropriate therapeutic strategy.

The invention is further described by the following examples, whereinall parts and percentages are by weight, and degrees are Celsius unlessotherwise stated.

EXEMPLIFICATION Example 1. Production of Chimeric Platelet SpecificIgG4.

A. General Strategy

The strategy for cloning the variable regions for the heavy and lightchain genes from the 7E3 hybridoma was based upon the linkage in thegenome between the variable region and the corresponding J (joining)region for functionally rearranged (and expressed) Ig genes. J regionDNA probes can be used to screen genomic libraries to isolate DNA linkedto the J regions; DNA in the germline configuration (unrearranged) wouldalso hybridize to J probes but is not linked to a variable regionsequence and can be identified by restriction enzyme analysis of theisolated clones.

The cloning strategy, therefore, was to isolate variable regions fromrearranged heavy and light chain genes using J_(H) and J_(K) probes.These clones were tested to see if their sequences were expressed in the7E3 hybridoma by Northern analysis. Those clones that containedexpressed sequences were put into expression vectors containing humanconstant regions and transfected into mouse myeloma cells to determineif an antibody was produced. The antibody from producing cells was thentested for binding specificity and functionality in comparison with the7E3 murine antibody.

A deposit of cell line derivative, murine hybridoma 7E3, was made withthe American Type Culture Collection, 12301 Parklawn Drive, Rockville,Md., 20852 on May 30, 1985. The accession number HB8832 was assignedafter successful viability testing.

B. Materials and Methods

Heavy Chain Genomic Library Construction

To isolate the heavy chain variable region gene from the 7E3 hybridoma,a size-selected genomic library was constructed using the phage lambdavector gt10. Southern analysis of Eco RI digested 7E3 DNA using a J_(H)probe revealed a single 3.5 kb band corresponding to a rearranged heavychain locus. It was likely that this fragment contained the 7E3 heavychain variable region gene. High molecular weight DNA was isolated from7E3 hybridoma cells and digested to completion with restrictionendonuclease Eco RI. The DNA was then fractionated on a 0.7% agarose geland the DNA of size range 3-4 kb was isolated directly from the gel.After phenol/chloroform extraction and fraction using a Sephadex® G-50gel filtration column, the 3-4 kb fragments were ligated with lambdagt10 arms (Promega Biotech, Inc) and packaged into phage particles invitro using Packagene® packing system from Promega Biotech. This librarywas screened directly at a density of approximately 30,000 plaques per150 mm petri dish using a ³² P-labeled J_(H) probe. Plaquehybridizations were carried out in 5× SSC, 50% formamide, 2× Denhardt'sreagent, 200 μg/ml denatured salmon sperm DNA at 42 degrees C. for 18-20hours. Final washes were in 0.5× SSC, 0.1% SDS at 65 degrees. Positiveclones were identified after autoradiography.

Light Chain Genomic Library Construction

To isolate the variable region gene for the 7E3 light chain, a genomiclibrary was constructed in the lambda vector EMBL-3. High molecularweight DNA was partially digested with restriction endonuclease Sau3Aand size-fractionated on a 10-40% sucrose density gradient. DNAfragments of 18-23kb were ligated with EMBL-3 arms and packaged intophage particles in vitro using Packagene® packaging system. This librarywas screened at a density of 30,000 plaques per 150 mm plate using aJ.sub.κ probe. Hybridization and wash conditions were identical to thoseused for the heavy chain library.

DNA Probes

The mouse heavy chain J_(H) probe is a 2 kb BamHI/EcoRI fragmentcontaining both J3 and J4 segments. The mouse light chain J.sub.κ probeis a 2.7 kb HindIII fragment containing all five J.sub.κ segments. ³²P-labeled probes were prepared by nick translation using a kit obtainedfrom Amersham, Inc. Free nucleotides were removed by centrifugationthrough a Sephadex® G-50 column. The specific activities of the probeswere approximately 10⁹ cpm/μg.

Northern Analysis

15 μg total cellular RNA was subjected to electrophoresis on 1%agarose/formaldehyde gels (Maniatis, et al, Molecular Cloning) andtransferred to nitrocellulose. Blots were hybridized with nicktranslated DNA probes in 50% formamide, 2× Denhardt's solution, 5× SSC,and 200 μg/ml denatured salmon sperm DNA at 42 degrees for 10 hours.Final wash conditions were 0.5× SSC 0.1% SDS at 65 degrees.

DNA Transfection using Electroporation

Plasmid DNA to be transfected was purified by centrifuging toequilibrium in ethidium bromide/cesium chloride gradients two times.10-50 μg of plasmid DNA was added to 8×10⁶ SP2/0 cells in PBS on ice andthe mixture placed in a Biorad electroporation apparatus.Electroporation was at 200 volts and the cells were plated out in 96well microtiter plates. Appropriate drug selection was applied after 48hours and drug resistant colonies were identified after 1-2 weeks.

Quantitation of Antibody Production

Tissue culture supernatant was analyzed for IgG protein content byparticle concentration fluorescence immunoassay (Jolley, M. E. et al,(1984) J. Immunol. Meth. 67:21) using standard curves generated withpurified IgG. Concentration of chimeric 7E3 antibody with human constantregions was determined using goat antihuman IgG Fc antibody-coatedpolystyrene beads and fluorescein conjugated goat anti-human IgG Fcantibody. The assay was carried out with an automated instrument (PandexLaboratories, Inc.)

Purification of Platelet-specific Chimeric IgG4 Antibody

Tissue culture supernatant was concentrated with a Diaflo YM100ultrafiltration membrane (Amicon), and loaded onto a proteinA-Sepharose® column. The chimeric antibody was eluted from the protein Acolumn with a sodium citrate pH gradient from pH 6.5 to pH 3.5. Thepurified antibody was concentrated using a Diaflo YM100 ultrafiltrationmembrane. Antibody concentration was measured by determining theabsorbance at 280 nm.

Binding Inhibition Assay

Purified antibody (either murine 7E3 or chimeric 7E3) was used tocompete with radioiodinated 7E3 antibody for binding to human platelets.Platelet-rich plasma (PRP) was prepared by centrifugation of citratedwhole human blood at 1875 rpm for 3.5 minutes. ¹²⁵ I-labeled 7E3antibody (150,000 cpm) was added to the appropriate dilution of thepurified test antibody and the reaction was initiated by the addition of150 μl PRP. Incubation was for 1-2 hours at room temperature and theplatelets with bound antibody were separated from free antibody bycentrifugation through 30% sucrose at 12,000 g for 4 minutes in a 0.4 mlmicrofuge tube. The tube tip containing the platelet/antibody pellet wascut off and counted in a gamma counter. The competition for binding toplatelets between iodinated 7E3 and chimeric 7E3 was compared to thecompetition between iodinated 7E3 and unlabeled 7E3 IgG.

Inhibition of Platelet Aggregation

Purified 7E3 or chimeric 7E3 antibody was added to citrated whole humanblood and incubated at 37 degrees for 10 minutes. The rate of plateletaggregation was measured after activation with collagen or ADP using awhole blood aggregometer (Chronolog Corp.).

C. Results

Cloning of the Platelet-specific Variable Gene Regions

Several positive clones were isolated from the heavy and light chainlibraries after screening approximately one million plaques using theJ_(H) and J.sub.κ probes, respectively. Following at least three roundsof plaque purification, bacteriophage DNA was isolated for each positiveclone, digested with either EcoRI (heavy chain clones) or HindIII (lightchain clones) and fractionated on 1% agarose gels. The DNA wastransferred to nitrocellulose and the blots were hybridized with J_(H)(heavy chain) or J.sub.κ ³² P-labeled DNA probes. For the heavy chain, 2clones were obtained that contained 3.5 kb Eco RI DNA fragments thathybridized to the J_(H) probe. Two size classes of HindIII fragments of3.0 and 6.0 kb were identified with the J.sub.κ probe.

Cloned DNA corresponding to the authentic heavy and light chain variableregions from the 7E3 hybridoma should hybridize to mRNA isolated fromthe hybridoma. Non-functional DNA rearrangements at either the heavy orlight chain loci should not be expressed. Northern analysis demonstratedthat the 3.5 kb EcoRI putative heavy chain fragment and the 6.0 kbHindIII putative light chain fragment each hybridizes to the appropriatesize mRNA from the 7E3 hybridoma. The subcloned fragments were labeledwith ³² P by nick translation and hybridized to northern blotscontaining total RNA derived from SP2/0 (the fusion partner of the 7E3hybridoma) or from 7E3, The 3.5 kb EcoRI heavy chain fragment hybridizedwith a 2 kb mRNA in 7E3 RNA but not in SP2/0 RNA. Similarly, the 6.0 kblight chain HindIII fragment hybridized with a 1250 bp mRNA in 7E3 RNAbut not in SP2/0 RNA. These are the correct sizes for heavy and lightchain mRNAs, respectively. Because the cloned DNA fragments containsequences expressed in the 7E3 hybridoma, these data suggest that theclones contain the correct variable region sequences from the 7E3hybridoma. The final functional test, however, is the demonstration thatthese sequences, when combined with appropriate constant regionsequences, are capable of directing the synthesis of an antibody with aspecificity and affinity similar to that of the murine 7E3 antibody.

Vectors and Expression Systems

The putative light and heavy chain V genes cloned from the 7E3 hybridomawere joined to human κ and G4 constant region genes in expressionvectors described previously (Sun, L. et al., Proc. Natl. Acad. Sci. USA84:214-218 (1987). The 17-1A V.sub.κ HindIII fragment ofpSV184ΔHneo17-1AV.sub.κ hC.sub.κ was replaced with the 6.0 kb HindlIIfragment corresponding to the putative light chain variable region genefrom 7E3. Similarly, the 17-1A V_(H) Eco RI fragment ofpSV2ΔHgpt17-1AV_(H) -hC_(G4) was replaced with the 3.5 kb EcoRI fragmentcorresponding to the putative heavy chain V region gene from 7E3. Thestructures of the resulting plasmids, designated p7E3V.sub.κ hC_(H)κ andp 7E3V_(H) hC_(G4), are shown in FIGS. 1A-1B.

To express the chimeric heavy and light chain genes, the two plasmidswere cotransfected into the nonproducing mouse myeloma cell line SP2/0.The light chain plasmid confers resistance to G418 and the heavy chainplasmid confers resistance to mycophenolic acid, thus allowing a doubleselection to be used to obtain clones carrying and expressing genes fromeach plasmid. Colonies resistant to G418 and mycophenolic acid wereexpanded to stable cell lines and maintained in the presence of bothdrugs. Tissue culture supernatant from these cell lines was tested forantibody using a particle concentration fluorescence immunoassay withpolystyrene beads coated with goat anti-human IgG Fc antibody and thesame antibody labeled with fluorescein. Out of the first 10 lineschecked, one (designated c-7E3F6) that produced approximately 2 μg/mlwas selected for further study.

Platelet Binding Activity Assay

After purification of c-7E3F6 antibody using a protein A-Sepharose®column, the antibody was concentrated and compared to murine 7E3 IgG inthe platelet binding activity assay. FIG. 2 shows that murine 7E3 andc-7E3F6 (the putative chimeric antibody) compete with radiolabeled 7E3for platelet binding to the same extent; the binding curves aresuperimposable indicating that the binding characteristics of murine andchimeric 7E3 are identical by this criterion.

Inhibition of Platelet Aggregation by c-7E3F6

Purified c-7E3F6 was compared to murine 7E3 in a functional assay thatmeasures the ability of the test antibody to inhibit aggregation ofhuman platelets. The results of such an assay are shown in FIG. 3 anddemonstrate that both antibodies inhibit collagen-induced plateletaggregation to the same extent at the same antibody concentration.c-7E3F6 also inhibits ADP-induced platelet aggregation to a similarextent.

The results of the platelet binding assay and the inhibition of plateletaggregation assay demonstrate that: (1) the correct variable regiongenes were indeed cloned from the 7E3 hybridoma; and (2) thesubstitution of the human constant regions for the murine constantregions has no effect on the binding or functional characteristics ofthe 7E3 variable regions as measured by these assays.

Fibrinogen-coated Bead Assay

The chimeric c-7E3F6 antibody was found positive in a qualitative,functional assay that measures the ability of an antibody to inhibit theagglutination between platelets and fibrinogencoated beads. Coller, B.et al. (1983) J. Clin. Invest. 73:325-338.

Example 2. Production of Chimeric IgGl and lgG3.

The DNA segment encoding the variable region of the heavy chain from themurine 7E3 antibody was linked to the human γ1 and γ3 constant regionspresent on the expression vectors pSV2ΔHgpt17-1AV_(H) -hC_(G1) andpSV2ΔHgpt17-1AV_(H) -hC_(G3) (Sun et al., Proc. Natl. Acad. Sci. USA84:214-218, 1987), by replacing the 17-1A heavy or light chain variableregion fragments with the corresponding 7E3 variable region fragments.The resulting chimeric heavy chain genes were cotransfected with thechimeric light chain gene into SP2/0 cells to generate stable cell linessecreting γ1,K, and γ3,K antibodies.

Example 3. Initial Studies of Use of Chimeric 7E3 Fab in Humans

Preparation of Chimeric 7E3 Fab Fragments

The Fab fragment of chimeric 7E3 (c7E3) was produced by enzymaticdigestion of purified chimeric 7E3 IgG (gamma 1 heavy chain, kappa lightchain) with the proteolytic enzyme papain. The Fab fragment was isolatedby a series of purification steps designed to yield a product which wasfree of other digestion fragments and other contaminating components(e.g., protein, nucleic acid, viruses). The final product was preparedas a sterile, non-pyrogenic solution containing 2 mg of monoclonalchimeric 7E3 Fab per ml of 0.15M sodium chloride, 0.01M sodiumphosphate, pH7.2. In certain preparations, polysorbate 80 was includedat a final concentration of 0.001% (w/v). Prior to use, the product wasfiltered through a 0.22 micron low protein binding filter. The productwas stored at 2°-8° C.

Pharmacokinetics: Plasma Clearance of c7E3 Fab in Humans

The plasma clearance of chimeric 7E3 (c7E3) Fab fragment was studied inthree patients with stable coronary disease. Following a 0.25-mg/kg doseof c7E3 Fab administered intravenously as a five minute infusion, bloodsamples were taken at various times from two minutes to 72 hours. It wasanticipated that a certain portion of the antibody would exist in anunbound state in plasma. To quantify this unbound antibody component, itwas necessary to rapidly separate the plasma from the platelets toprevent further binding ex vivo. The plasma concentration of free c7E3Fab was measured by solid-phase enzyme immunoassay (EIA). The assayemployed affinity isolated anti-murine 7E3 IgG purified from rabbitantisera for solid-phase capture and a detection system based on abiotinylated derivative of the same rabbit anti-7E3 antibodypreparation. The results are presented in Table 1.

                  TABLE 1    ______________________________________    PLASMA CONCENTRATION OF c7E3 Fab IN PATIENTS    TREATED WITH A 0.25-MG/KG DOSE           c7E3 Fab (μg/mL)    Time*    Patient A    Patient B                                   Patient C    ______________________________________    Pre-dose ND           ND       ND    2     min    NA           2.554  2.312    5     min    1.149        1.873  1.411    10    min    0.714        1.331  1.111    15    min    0.610        0.916  0.852    20    min    0.499        0.985  0.756    30    min.   0.464        0.815  0.515    45    min    0.340        0.704  0.405    1     hr     0.309        0.437  0.195    2     hr     0.288        0.262  0.149    6     hr     0.204        0.095  0.105    12    hr     0.112        0.072  0.064    24    hr     0.065        0.058  0.046    48    hr     0.055        1.47   0.175    72    hr     0.196        ND     0.076    ______________________________________     *Interval between end of infusion and blood drawing. Note that platelets     were in contact with the plasma for an additional 2 minutes after the     blood was drawn (i.e., for the time required to separate the plasma by     centrifugation).     ND = Not detected/below the detectable level of the assay (0.025     μg/mL).     NA = Not available.

If the entire injected dose of 7E3 were detected as free antibody inplasma, the theoretical maximum antibody concentration would beapproximately 6.25 μg/mL (0.25 mg/kg divided by 40 mL of plasma/kg).However, this theoretical maximum concentration would never be attainedbecause of the large component of injected antibody which binds toplatelets. In fact, at the earliest measurement time (2 minutes), theaverage plasma concentration (n=2) of c7E3 Fab was 2.43 μg/mL; thisvalue was the observed maximum plasma concentration (C_(max)). The dataobtained at subsequent post-injection times show a rapid initialdecrease in the plasma concentration of c7E3 Fab. By the 1-hour and24-hour measurements, the administered antibody remaining in the plasma(n=3) was less than 0.5 μg/mL and 0.1 μg/mL, respectively. A plot of theplasma antibody concentration (ng/mL) versus time data, presented inFIG. 4 graphically demonstrates the rapid initial clearance of c7E3 Fabfrom the plasma in all three patients.

A preliminary analysis of the pharmacokinetic characteristics of c7E3Fab fragment was undertaken. Several models, including several mixed(random and fixed effects) linear models as well as standardtwo-compartmental and non-compartmental models, were used to fit theplasma concentration data. The free plasma antibody data did notadequately fit standard pharmacokinetic models. As the site of actionfor c7E3 Fab is a receptor located on platelets, it is not unexpectedthat the plasma concentration of free antibody would not be related toits concentration at its site of action in any simple way. The rapidinitial clearance of c7E3 Fab from the plasma reflects, in part, therapid antibody binding to platelet GPIIb/IIIa receptors. Of the modelsexamined, the random effects linear model was shown to best fit theplasma concentration data. Using this model, preliminary values for thepharmacokinetic parameters, Cl_(p), V_(d), and t_(1/2), were determinedand are presented in Table 2.

                  TABLE 2    ______________________________________    PHARMACOKINETIC VALUES FOR c7E3 Fab*    Parameter             Value    ______________________________________    Cl.sub.p (proportion/hr)                          15.6    V.sub.d (L)            6.8    t.sub.1/2  (hr)        0.1 (6 min)    ______________________________________     *A random effects linear model was used to fit the data.     Cl.sub.p = Plasma clearance is defined as the rate of decrease in plasma     concentration divided by the concentration and is computed as a rate per     hour, i.e., if the rate at a given time continued for an hour, the     computed rate would be the proportion of drug removed in that hour.     V.sub.d = Volume of distribution is defined as the dose administered     divided by the measured plasma concentration multiplied by the plasma     volume. A 3L plasma volume typical for a 70kg person was used in the     calculations.     t.sub.1/2  = Elimination halflife.

Urinary Excretion in Humans

Urine samples were collected from three patients with stable coronarydisease who were treated intravenously with 0.25-mg/kg of c7E3 Fab(plasma clearance data for these same three patients are discussedabove). Total urine output was collected for the followingpost-injection time periods: 0 to 2 hours, 2 to 6 hours, 6 to 12 hours,and 12 to 24 hours. In addition, a sample of predose urine was alsocollected. Representative samples of the collected urine samples wereanalyzed for free 7E3 Fab using a slight modification of the EIAdescribed above. In all cases, no c7E3 Fab was detected in the urine.

Preclinical Toxicology

Preclinical toxicology studies have been performed in 18 monkeys(Cyonomolgus and Rhesus), using chimeric 7E3 Fab. Bolus doses of up to0.6 mg/kg, followed by infusion of up to 0.8 μg/kg/min for 96 hours wereadministered (includes studies with heparin, aspirin and recombinanttissue plasminogen activator). In all monkeys, at all doses, in allcombinations, 7E3 was safe and well-tolerated, with no significantbleeding complications or other adverse events.

Dose Escalation of Chimeric 7E3 Fab in Stable Angina Patients A doseescalation study was conducted enrolling 52 stable angina patients(males from 43 to 75 years old) who were off anti-platelet therapy formore than 10 days. A variety of dosing regimens were administered.Patients received either single intravenous bolus injections of 0.15 to0.30 mg/kg of chimeric 7E3 Fab (20 patients) or a bolus loading followedby continuous intravenous infusions (10 μg/minute) from 12 to 96 hoursin duration (32 patients).

Platelet GPIIb/IIIa receptor blockade, platelet aggregation in responseto 20 μM ADP (agonist), and bleeding times were determined 2 hours afteradministration of a bolus dose of c7E3 Fab (0.15-0.30 mg/kg). Receptorblockade and platelet aggregation in response to agonist were determinedessentially as described (Gold, H. K. et al., J. Clin. Invest., 86:651-659 (1990)). Bleeding times were determined by the Simplate method.With increasing doses there was a progressive increase in receptorblockade, as indicated by the percent of receptors blocked (determinedfrom the availability of receptor binding sites). The increase inreceptor blockade was paralleled by inhibition of platelet aggregation(measured as a percent of the pre-dose value or baseline), and by anincrease in bleeding time.

The peak effect in terms of all three parameters was observed at 0.25mg/kg. This dose corresponds to a plasma concentration of 5 μg/ml--theconcentration at which peak inhibition was seen in a platelet-richplasma from a normal subject which had been incubated for 15 minutes inan aggregometer cuvette in the presence of increasing concentrations ofchimeric 7E3 Fab. (The extent of aggregation of the plasma of the normalsubject was measured by the percent of light transmitted through thecuvette. Prior to the addition of an agonist, the plasma was relativelyopaque and the percent of light transmitted was set at zero. When theagonist ADP was added to a control sample without antibody, the lighttransmission progressively increased as aggregation progressed. However,when c7E3 Fab is present, a dose-dependent block of aggregation wasobserved with complete inhibition at 5 μg/ml c7E3 Fab.)

The duration of action in terms of receptor blockade, inhibition ofplatelet aggregation, and bleeding time was determined. Peak effects onreceptor blockade, platelet agregation, and bleeding time were seen at 2hours, with gradual recovery over time. Bleeding times returned to nearnormal values by 6-12 hours.

Because peak receptor blockade and functional inhibition were achievedwith 0.25 mg/kg, the duration of platelet inhibition by continuousinfusions following this loading dose were assayed to determine if theduration of platelet inhibition could be prolonged. The degree ofreceptor blockade, inhibition of platelet aggregation, and prolongationof bleeding time were maintained for the duration of continuous infusionin five patients who received a 10 μg/minute continuous infusion ofchimeric 7E3 Fab for 72 hours following the 0.25 mg/kg loading dose.Recovery started as soon as the infusion was discontinued. Similarresults were seen with 12, 24, 48 and 96 hour infusions.

None of the patients experienced a hypersensitivity reaction. There wereno significant treatment related trends in hematology or chemistrylaboratory values. Nor were there any major bleeding events.Insignificant bleeding events were rare and included transient mild nosebleed and mild gum oozing in patients with periodontal disease. Theresults of the trial indicated that chimeric 7E3 Fab can be administeredto patients using dosing regimens that produce profound inhibition ofplatelet function for periods as long as several days.

Immunogenicity Results

In trials with murine 7E3 F(ab')₂ and Fab (150 patients), immuneresponses detected using a sensitive enzyme-linked immunoassay systemoccurred in 16% (24/150) of the patients. All reactions were of lowtiter, typically in the range of 1:50 to 1:200. The treatment groupincluded normal volunteers treated with 0.01-0.25 mg/kg murine 7E3F(ab')₂, unstable angina patients treated with 0.05-0.20 mg/kg murine7E3 F(ab')₂, and PTCA patients treated with 0.1 mg/kg murine 7E3 F(ab')or 0.15-0.35 mg/kg murine Fab, as well as stable angina patients treatedwith a single bolus intravenous injection of 0.10-0.30 mg/kg of murine7E3 Fab, a single bolus dose of either 0.25 or 0.30 mg/kg followed bycontinuous infusion for 12-36 hours (0.15 μg/kg/min or 10 μg/min) ofmurine Fab, or with two injections of murine Fab separated by six hours(a single bolus of 0.2 mg/kg-0.30 mg/kg followed by a bolus of 0.05mg/kg).

Immunogenicity was notably reduced with the human-mouse chimeric 7E3Fab. None of the 52 patients having stable angina enrolled in the doseescalation study and treated with chimeric 7E3 Fab (see above) showed animmune response to treatment as measured by a similar assay adapted tothe chimeric Fab.

Reversibility of Anti-platelet Activity

Chimeric 7E3 Fab (γ₁, κ) has a slow off rate from platelets and freeplasma chimeric 7E3 Fab clears from circulation rapidly (see above).Thus, the antiplatelet effects of chimeric 7E3 are readily reversible byadministration of random donor platelets. This reversal or antidoteeffect by transfusion of platelets has been demonstrated in 2 patientswho had received either murine Fab or chimeric Fab and who receivedrandom donor platelets during a time when they had nearly completeinhibition of platlet aggregation. Restoration of platelet function wasdetermined by measuring bleeding times. This property is useful insitutations where a bleeding event necessitates restoration of plateletfunction in a patient.

Example 4. Use of Chimeric 7E3 Antibody in the Prevention of ThromboticComplications of Elective Coronary Angioplasty.

Percutaneous transluminal coronary angioplasty (PTCA), by balloon orcoronary atherectomy, for example, is an effective method of enlargingthe lumen of stenosed coronary arteries. In this procedure, there is aninherent risk of acute coronary occulusion during and after angioplasty.The reported rate of coronary occlusion varies from approximately 3%-6%of elective angioplasty cases (Detre, K. M. et al., Circulation 82:739-750 (1991)), and is the major cause of in-hospital morbidity andmortality. In high risk patients, the incidence of major cardiac eventscaused by thrombosis is between 10-20%.

Acute coronary occlusion during or immediately after coronaryangioplasty appears to be caused by the combination of deep arterialwall injury with resultant partially occlusive "intimal flaps" with orwithout superimposed thrombus formation, or thrombus formation alone ata site of vessel wall injury. In animal models, reocclusion aftersuccessful thrombolysis is preceded by periods of cyclical reductionsand restorations in coronary blood flow termed "cyclic flow variations"(CFVs). These CFVs are almost entirely a platelet-mediated phenomenon,and are due to repetitive accumulation and dislodgement of plateletaggregates at sites of coronary stenosis and endothelial injury. Cyclicflow variations after coronary angioplasty have been described inhumans. Chimeric 7E3 antibody can be used to inhibit platelet functionduring angioplasty thereby preventing platelet aggregation andthrombosis. Chimeric 7E3 antibody is particularly useful in patients athigh risk of thrombotic occlusion. These patients can be identified onthe basis of anatomic (e.g., angiographically defined characteristics ofa lesion at a site of stenosis) or clinical risk factors (e.g.,myocardial infarction, unstable angina, diabetes, women 65 years orolder), which predispose to acute coronary thrombosis and produce theclinical syndromes of acute myocardial infarction, unstable angina orabrupt closure.

Chimeric Anti-platelet Antibody in Elective PTCA

The trial was conducted in two stages. The primary objective of thefirst stage was to determine the safety and optimal dose of single dosechimeric 7E3 Fab in patients undergoing elective percutaneoustransluminal coronary angioplasy (PTCA). Stage II was conducted toevaluate the safety and preliminary efficacy of chimeric 7E3 (c7E3) whenadministered by bolus infusion followed by various continuous infusiondurations. The Stage II study comprised elective coronary angioplastypatients who were at risk for ischemic cardiac complications. High riskpatients included those with unstable angina or stable coronary diseasewith Type B or C lesion specific characteristics. Table 3 lists thedefinitional criteria for high risk patients, and Table 4 lists theangiographically defined lesion-specific characteristics. Preliminaryefficacy was measured as inhibiton of platelet function and preventionof thrombotic complications. Men, between 18 and 76 years of age, andwomen not of child bearing potential, between 18 and 76 years of age,were eligible to enroll in both stages of the trial.

                  TABLE 3    ______________________________________    ENROLLMENT CRITERIA FOR PATIENTS AT HIGH RISK FOR    ISCHEMIC COMPLICATIONS    ______________________________________    Moderately high risk:    1)       Unstable angina with no lesion-specific             characteristic defined.    2)       A stenosis with a single Type B lesion-specific             characteristic.    Highest risk:    1)       A stenosis with ≧ two Type B lesion-specific             characteristics    2)       Unstable angina with a stenosis with at least             one Type B lesion-specific characteristic.    3)       Diabetes mellitus with a stenosis with at least             one Type B lesion-specific characteristic.    4)       Women ≧ 65 years of age with a stenosis with at             least one Type B lesion-specific characteristic.    5)       A stenosis with at least one Type C             lesion-specific characteristic.    ______________________________________

                  TABLE 4    ______________________________________    LESION-SPECIFIC CHARACTERISTICS    ______________________________________    Type A Lesions (high success, > 85%; low risk)    Discrete (< 10 mm length)                     Little or no calcification    Concentric       Less than totally occlusive    Readily accessible                     Not ostial in location    Nonangulated segment, <45°                     No major branch involvement    Smooth contour   Absence of thrombus    Type B Lesions (moderate success, 60-85%; moderate risk)    Tubular (10-20 mm length)                     Moderate to heavy calcification    Eccentric        Total occlusions <3 months old    Moderate tortuosity of                     Ostial in location    proximal segment    Moderately angulated                     Bifurcation lesions requiring    segment, >45°, <90°                     double guide wires    Irregular contour                     Some thrombus present    Type C Lesions (low success, < 60%; high risk)    Diffuse          Total occlusion > 3 months old    Excessive tortuosity of                     Inability to protect major    proximal segment side branches    Extremely angulated                     Degenerated vein grafts with    segments > 90°                     friable lesions    ______________________________________

Stage I

In Stage I, patients were enrolled in groups receiving a single bolusintravenous injection of chimeric 7E3 (γ₁, κ) Fab fragment (prepared andformulated as described in Example 3). A total of 15 patients, (3 womenand 12 men) were treated. The median age of patients was 62 years (range46 years to 76 years). A demographic profile is listed in Tables 5A and5B for all single dose patients and for patients within the individualdose groups.

Five patients (n=5) each received single doses of 0.15 mg/kg, 0.20 mg/kgor 0.25 mg/kg of c7E3 Fab within about 30 minutes prior to elective PTCAin a dose-escalation protocol. All patients were treated with aspirin(standard dose) and fully anticoagulated with heparin (standard dose) atthe time of the procedure.

Although the PTCA procedures were classified as elective for Stage Ipatients, six of the 15 patients had unstable rest angina. The coronarylocation of the dilatations is summarized in Table 6 (bottom). Seven ofthe 15 Stage I patients underwent PTCA of one lesion in a single vessel,6 underwent multi-lesion PTCA in a single vessel, and 2 patients hadmulti-vessel PTCA performed (Table 6).

The efficacy criteria for obtaining the optimal single dose of c7E3 wereprospectively defined as the minimum dose that achieved median values ofthe following at 2 hours post-infusion: (1) prolongation of bleedingtime of at least 20 minutes; (2) blockade of GPIIb/IIIa receptors suchthat there were greater than 80% of baseline receptor sites blocked; and(3) an inhibition of platelet aggregation in response to 20 μM ADP to≦20% of baseline.

                  TABLE 5A    ______________________________________    CHIMERIC 7E3 ANTI-PLATELET ANTIBODY    Patient Classification of Age, Weight, Height, Sex and Race               Continuous                         Control Single Dose    ______________________________________    TOTAL PATIENTS                 32          9       15    AGE    Mean         57.4        54.2    60.1    Median       57.0        56.0    62.0    Minimum      38          37      46    Maximum      76          74      76    Std. Dev.    9.7         10.5    9.7    WEIGHT (kg)    Mean         82.8        88.2    85.5    Median       84.8        84.2    84.0    Minimum      42.3        67.3    70.5    Maximum      113.0       122.7   107.0    Std. Dev.    16.6        19.0    11.1    HEIGHT (cm)    Mean         171.2       176.7   173.6    Median       172.8       177.8   175.2    Minimum      152.4       157.5   160.0    Maximum      185.0       185.4   188.0    Std. Dev.    7.9         8.8     7.8    SEX    Female       8           1       3    Male         24          8       12    RACE         0           0       1    White        26          8       11    Black        5           1       2    Asian        0           0       1    Hispanic     1           0       0    ______________________________________

                                      TABLE 5B    __________________________________________________________________________    CHIMERIC 7E3 ANTI-PLATELET ANTIBODY    Patient Classification of Age, Weight, Height, Sex and Race                               0.25 mg/kg                                     0.25 mg/kg                                           0.25 mg/kg                               7E3*  7E3*  7E3*             0.15 mg/kg                   0.20 mg/kg                         0.25 mg/kg                               10 mcg/min                                     10 mcg/min                                           10 mcg/min             7E3   7E3   7E3   for 6 hrs                                     for 12 hrs                                           for 24 hrs                                                 Placebo    __________________________________________________________________________    TOTAL PATIENTS             5     5     5     11    11    10    9    AGE    Mean     60.6  57.2  62.6  59.1  55.0  58.1  54.2    Median   63.0  56.0  65.0  60.0  53.0  58.5  58.0    Minimum  47    46    51    40    42    38    37    Maximum  73    68    76    76    73    75    74    Std. Dev.             10.5  8.3   11.3  10.5  8.9   10.3  10.5    WEIGHT (kg)    Mean     83.8  80.5  92.2  85.8  83.8  78.3  88.2    Median   76.3  78.0  96.0  89.0  85.0  79.9  84.2    Minimum  74.5  70.5  82.0  60.4  42.3  62.3  67.3    Maximum  107.0 95.0  99.1  113.0 111.8 95.0  122.7    Std. Dev.             13.8  9.5   7.7   17.8  19.6  10.7  19.0    HEIGHT (cm)    Mean     170.8 171.9 178.1 169.3 170.9 173.7 176.7    Median   173.0 175.2 177.8 167.6 172.7 175.2 177.8    Minimum  160.0 160.8 169.0 157.5 152.4 165.1 157.5    Maximum  177.8 182.9 188.0 177.8 185.0 180.3 185.4    Std. Dev.             7.0   8.8   7.0   6.4   10.8  5.4   8.8    SEX    Female   3     0     0     3     4     1     1    Male     2     5     5     8     7     9     8    RACE             0     0     1     0     0     0     0    White    3     4     4     8     9     9     8    Black    2     0     0     3     2     0     1    Asian    0     1     0     0     0     0     0    Hispanic 0     0     0     0     0     1     0    __________________________________________________________________________

                  TABLE 6    ______________________________________    ANGIOGRAPHIC CHARACTERISTICS                          c7E3 Fab  c7E3 Fab                   Control                          Stage I   Stage II    ______________________________________    Number of Lesions Attempted:    Single vessel/single lesion                     8        7         21    Single vessel/multilesion                     0        6          7    Multivessel/single lesion                     1        2          1    Multivessel/multilesion                     0        0          2    Unknown          0        0          1    Location of Attempted Lesions*:    RCA              3        9         13    LCX              4        3         14    LAD              3        9         17    ______________________________________     *In patients with multivessel disease, both vessels are counted.     RCA = Right coronary artery     LCX = Left circumflex coronary artery     LAD = Left anterior descending coronary artery

Stage II

In Stage II, patients were treated with a 0.25 mg/kg bolus dose followedby a continuous infusion of 10 μg/min of c7E3 Fab for 6, 12, or 24hours. A total of 32 patients (8 women and 24 men) were entered into thetreatment group of Stage II of the study. The median age of the c7E3Fab-treated patients was 57 years (range 38-76). Nine control patients(1 woman, 8 men) were entered. The median age of control patients was 56years (range 37-74). Control patients were high risk patients as definedabove, who did not receive c7E3, but were monitored and followed in thesame fashion as treated patients. A demographic profile for all Stage IIpatients and for patients within the individual dose groups is listed inTables 5A and 5B.

Treatment with c7E3 Fab was initiated 30 minutes prior to ballooninflation for PCTA. Aspirin and heparin were given as clinicallyindicated, with the recommendation that following angioplasty heparin begiven at the rate of 800 units per hour. Eleven patients each wereentered into the 6 and 12 hour groups, and ten patients were enteredinto the 24 hour group.

Of the 32 c7E3 Fab-treated patients, 21 patients underwent PTCA of onelesion in a single vessel, 7 patients underwent multi-lesion PTCA in asingle vessel, and 3 patients had multi-vessel PTCA performed (Table 6).The type of PTCA was not specified in one patient. The coronary locationof the dilatations for stage II patients is summarized in Table 6. Ofthe 9 control patients, 8 underwent PTCA of one lesion in a singlevessel, and one patient had multi-vessel PTCA. The 32 c7E3 Fab-treatedpatients and the 9 control patients had clinical or angiographiccharacteristics that would classify them as high risk for ischemiccardiac complications of PTCA. Two c7E3 Fab-treated patients and onecontrol patient had unspecified risk factors. The remaining 30 c7E3Fab-treated patients and 8 contol patients had at least one identifiableclinical feature or angiographic characteristic placing them atincreased risk of ischemic complication, and most had more than one riskfactor. Table 7 summarizes these risk factors for the control and c7E3treatment groups, and individual listings of risk factors by patientwithin each dose group are presented in Tables 8A through 8D.

                  TABLE 7    ______________________________________    HIGH RISK CHARACTERISTICS    Stage II                      Control c7E3 Treatment    Risk Factors      (n = 9) (n = 32)    ______________________________________    One Type B characteristic                      .sup. 5.sup.1                              4.sup.    Two or more Type B                      .sup. 1.sup.2                              7.sup.3    characteristics    One Type C characteristic                      0       2.sup.    Unstable angina with no                      1       1.sup.4    lesion characteristics    identified    Unstable angina + 0       8.sup.5    Type B characteristic    Unstable angina + ≧ 2                      1       7.sup.6    Type B characteristics    Unstable angina + 0       1.sup.    Type C characteristic    Unspecified risk  1       2.sup.    characteristic    ______________________________________     .sup.1 Patient 04006 had diabetes     .sup.2 This patient (04007) had diabetes     .sup.3 Patients 03001 and 02007 had diabetes     .sup.4 This patient (04004) had the following additional risk factors:     female, age > 65, and diabetes     .sup.5 Patients 03003 and 05001 had diabetes     .sup.6 Patient 01018 had the following additional risk factors: female an     age > 65. Patient 03002 had diabetes.

                  TABLE 8A    ______________________________________    PREDISPOSING HIGH RISK CHARACTERISTICS    Control Patients    Patient    Number   Type of Risk(s).sup.1 Segment.sup.2    ______________________________________    01-023   1.    Unstable angina     LAD    01-024   1.    One Type B charasteristic.sup.3                                       LAD    04-006   1.    Diabetes            LCX             2.    One Type B characteristic                   (eccentric)    04-007   1.    Diabetes            RCA             2.    Two Type B characteristics                   (eccentric; moderately                   angulated segment; > 45°, < 90°)    04-008   1.    One Type B characteristic                                       LCX                   (eccentric)    04-009   1.    Our Type B characteristic                                       LCX                   (eccentric)    01-021   1.    One Type B characteristic.sup.3                                       RCA    01-022   1.    Unstable angina     OM             2.    Two Type B characteristics                   (tubular; irregular contour)    03-005   1.    Unspecified risk    RCA                   characteristic    ______________________________________     .sup.1 Potential characteristics listed in Tables 3 and 4     .sup.2 RCA = right coronary artery     LCX = left circumflex coronary artery     LAD = left anterior descending coronary artery     OM = obtuse marginal branches of LCX     .sup.3 Characteristic not designated

                  TABLE 8B    ______________________________________    PREDISPOSING HIGH RISK CHARACTERISTICS    6-Hour Continuous Infusions    Patient    Number   Type of Risk(s).sup.1                                  Segment.sup.2    ______________________________________    04-001   1.    Unstable angina    RCA             2.    One Type B characteristic                   (eccentric)    06-001   1.    Unstable angina    LCX             2.    One Type B charasteristic                   (thrombus)    06-002   1.    One Type B characteristic                                      LAD                   (tubular  10 to 20 mm lesion!)    01-014   1.    Unstable angina    LAD             2.    One Type C characteristic                   (Diffuse > 2 cm length)    01-013   1.    Unstable angina    RCA             2.    Two Type B characteristics                   (eccentric, some thrombus)    01-015   1.    Two or more Type B LAD,                   characteristics    LADD    01-017   1.    One Type B characteristic                                      LCX                   (eccentric)    02-005   1.    Unstable angina    LAD             2.    Three Type B characteristics                   (tubular, (10 to 20 mm length);                   irregular contour; ostial in                   location)    ______________________________________     .sup.1 Potential characteristics listed in Tables 3 and 4     .sup.2 RCA = right coronary artery     LCX = left circumflex coronary artery     LAD = left anterior descending coronary artery     LADD = diagonal branch of LAD     .sup.3 Characteristic not designated    Patient    Number   Type of Risk(s).sup.1                                  Segment.sup.2    ______________________________________    03-001   1.    Diabetes           LAD             2.    Four Type B characteristics                   (eccentric; moderate angulation,                   >45°, <90°; irregular contour;                   moderate to heavy calcification)    01-012   1.    One Type C characteristic                                      LAD                   (Diffuse >2 cm length)    01-016   1.    One Type B characteristic                                      RCA                   (some thrombus)    OM.sub.n    ______________________________________     .sup.1 Potential characteristics listed in Tables 3 and 4     .sup.2 RCA = right coronary artery     LCX = left circumflex coronary artery     LAD = left anterior descending coronary artery     OM.sub.n = obtuse marginal branches of LCX

                  TABLE 8C    ______________________________________    PREDISPOSING HIGH RISK CHARACTERISTICS    12-Hour Continuous Infusion    Patient Number              Type of Risk(s).sup.1                                   Segment.sup.2    ______________________________________    01-018    1.    Unstable angina    OM.sub.n              2.    Female over 65              3.    Two or more Type B                    characteristics    01-019    1.    Unstable angina    Circumflex              2.    Two or rnore Type B                    characteristics    02-006    1.    Unstable angina    RCA              2.    One Type B characteristic                    (total occlusion <3 months old)    02-007    1     Diabetes           LCX              2.    Five Type B characteristics                                       OM.sub.1, LADD                    (eccentric, moderate tortuosity                    of proximal segment; moderately                    angulated segment, >45°, <90°;                    bifurcation lesions requiring                    double guidewires; total                    occlusions <3 mo)    03-002    1.    Unstable angina    LAD              2.    Diabetes              3.    Two Type B characteristics                    (moderate tortuosity segment;                    irregular contour)    03-003    1.    Unstable angina    RCA              2.    Diabetes              3.    One Type B characteristic                    (irregular contour)    ______________________________________     .sup.1 Potential characteristics listed in Tables 3 and 4     .sup.2 RCA = right coronary artery     LCX = left circumflex coronary artery     LAD = left anterior descending coronary artery     OM.sub.n = obtuse marginal branches of LCX    Patient Number              Type of Risk(s).sup.1                                   Segment.sup.2    ______________________________________    05-001    1.    Unstable angina    LADD              2.    Diabetes           RCA              3.    One Type B characteristic                    (eccentric)    05-002    1.    One Type B characteristic                                       RCA                    (tubular)              2.    One Type C characteristic                                       LADD                    (total occlusion >3 months)    05-003    1.    Two Type B characteristics                                       LAD                    (moderately angulated segment,                    >45°, <90°; some thrombus)    06-003    1.    Unstable angina    RCA              2.    One Type B characteristic                    (some thrombus)    04-002    1.    Unstable angina    LCX              2.    One Type B characteristic                    (tubular 10 to 20 mm)    ______________________________________     .sup.1 Potential characteristics listed in Tables 3 and 4     .sup.2 RCA = right coronary artery     LCX = left circumflex coronary artery     LAD = left anterior descending coronary artery     LADD = diagonal branch of LAD     OM = obtuse marginal branches of LCX     .sup.3 Characteristic not designated

                  TABLE 8D    ______________________________________    PREDISPOSING HIGH RISK CHARACTERISTICS    24-Hour Continuous Infusion    Patient    Number    Type of Risk(s).sup.1                                  Segment.sup.2    ______________________________________    01-020    1.    Two Type B characteristics                                      RCA                    (irregular contour, some                    thrombus)    02-008    1.    Unstable angina              2.    Type B characteristics              a)  4 characteristics                                  LCX                  (tubular; eccentric                  moderate tortuosity                  of proximal segment;                  irregular contour)              b)  3 characteristics                                  LCX                  (eccentric; moderately                  angulated segment >45°,                  <90°)    05-005    1.    Unstable angina   RCA              2.    One Type B characteristic.sup.3    05-006    1     Unstable angina   RCA              2.    One Type C characteristic                    (diffuse (>2 cm length))    04-003    1.    Two Type B characteristics                                      LAD                    (eccentric; bifurcation                    with double guidewires)    04-004    1.    Unstable angina   LAD              2.    Diabetes              3.    Female over 65    ______________________________________     .sup.1 Potential characteristics listed in Tables 3 and 4     .sup.2 RCA = right coronary artery     LCX = left circumflex coronary artery     LAD = left anterior descending coronary artery     LADD = diagonal branch of LAD     OM.sub.n = obtuse marginal branches of LCX     .sup.3 Characteristic not designated    Patient    Number    Type of Risk(s).sup.1                                  Segment.sup.2    ______________________________________    04-005    1.    Two Type B characteristics                                      RCA                    (eccentric, some thrombus)    02-009    1.    Unstable angina              2.    Type B characteristics              a)  3 characteristics                                  LAD                  (irregular contour, some                  thrombus; bifurcation                  lesions requiring double                  guidewires)              b)  1 characteristic                                  LAD                  (bifurcation lesions re-                  quiring double guidewires)              c)  2 characteristics                                  DB                  (eccentric; bifurcation                  lesions requiring double                  guidewires)    05-004    1.    One Type B characteristic                                      OM.sub.1                    (bifurcation requiring double                                      OM.sub.2                    guidewires)    ______________________________________     .sup.1 Potential characteristics listed in Tables 3 and 4     .sup.2 RCA = riqht coronary artery     LCX = left circumflex coronary artery     LAD = left anterior descending coronary artery     DB     OM.sub.n = obtuse marginal branches of LCX     .sup.3 Characteristic not designated

Inhibition of Platelet Function (Stage I Results)

To assess activity of the chimeric 7E3 Fab in inhibiting plateletfunction, GPIIb/IIIa receptor binding site availability (recorded asmedian percent GPIIb/IIIa blocked), median inhibition of agonist-inducedplatelet aggregation in response to 20 μM ADP, and median bleedingtimes, were serially measured. Receptor blockade and plateletaggregation in response to agonist were determined essentially asdescribed (Gold, H. K. et al., J. Clin. Invest., 86: 651-659 (1990)).For receptor blockade measurements, receptor availability was measuredat time 0 and the number of receptors available were taken as 0%receptors blocked (baseline). Other time points are relative to thenumber of receptors available at baseline or the pre-dose measurement.Bleeding times were determined by the Simplate method.

FIGS. 5A-5C summarizes the dose response 2 hours following a singlebolus dose of chimeric 7E3 Fab, in terms of receptor blockade (FIG. 5A),platelet aggregation (FIG. 5B), and bleeding time (FIG. 5C). The solidlines in FIGS. 5A-5C indicate the median values of the 5 patientsstudied at each dose group. With increasing doses of c7E3 Fab there wasa progressive increase in receptor blockade as shown in percent ofreceptors that are blocked (FIG. 5A). The median number of receptorsblocked at two hours was 53.8% for the 0.15 mg/kg, 80.2% for the 0.20mg/kg, and 86.6% for the 0.25 mg/kg dose groups. The increase inreceptor blockade was paralleled by inhibition of platelet aggregation,depicted as a percent of the pre-dose value (FIG. 5B). Median plateletaggregation at 2 hours was 46.1%, 44.6%, 17.9% of baseline for the 0.15mg/kg, 0.20 mg/kg, and 0.25 mg/kg dose groups, respectively. Likewise, adose-related prolongation of bleeding time was seen at 2 hourspost-infusion (bleeding time measurements were truncated at 30 minutes;FIG. 5C). The median bleeding times were 26.0 minutes, 27.5 minutes, and30 minutes for the 0.15 mg/kg, 0.20 mg/kg, and 0.25 mg/kg doses,respectively. Under the conditions used, and as measured by theseassays, the optimal dose for anti-platelet activity was determined to be0.25 mg/kg.

FIGS. 6A-6C show the duration of action following a single bolus dose of0.25 mg/kg, the dose at which maximum platelet effects were seen. Thelines indicate the median values from time zero (baseline) through 24hours, as shown on the x-axis, in terms of receptor blockade in the toppanel (FIG. 6A), platelet aggregation in the middle panel (FIG. 6B), andbleeding time in the bottom panel (FIG. 6C). Peak effects on receptorblockade, platelet aggregation, and bleeding time are seen at 2 hours,with gradual recovery over time. Bleeding times return to near normalvalues by 12 hours. None of the patients experienced thrombocytopenia.

Inhibition of Platelet Function (Stage II Results)

In Stage II, GPIIb/IIIa receptor and platelet aggregation data were notobtained in all patients, and only two patients in the 24 hour infusionhad these studies performed. Therefore, only the 6 and 12 hour data aresummarized. In both the 6-hour and 12-hour infusion groups, medianreceptor blockade was maintained to greater than 80% of baseline throughthe duration of infusion. Median 20 μM ADP-induced platelet aggregationfor the 6 and 12-hour infusion groups was 13% and 15% of baseline at 2hours, respectively, and in the 12 hour group remained below 25% for theduration of the infusion. The 2-hour median bleeding time for all threeinfusion durations was greater than 30 minutes. FIGS. 7A-7C review theresults observed in patients who received a 10 μg/minute continuousinfusion of chimeric 7E3 Fab for 12 hours following the 0.25 mg/kgloading dose. The lines represent the median values. The degree ofreceptor blockade, inhibition of platelet aggregation, and prolongationof bleeding time are maintained for the entire duration of the infusion,with recovery starting as soon as the infusion is discontinued.

                  TABLE 9    ______________________________________    MEDIAN DATA FROM FIGS. 7A-7C    0.25 mg/kg + μg/min for 12 hours    Time following               Median                  Median    On-set of infusion               Bleeding Time                          Median Aggregation                                       Binding    Hours      Minutes    % Baseline   % Baseline    ______________________________________     0         5.5        100           0.0     2         30         14.7         93.5     6         30         22.4         89.1    12         23.5       24.4         85.6    18         13.9       61.1         72.9    24         8.6        60.9         69.2    36         14.5       75.0         60.6    ______________________________________

Clinical Outcomes of Stage I and Stage II Patients

None of the 47 c7E3 Fab-treated patients experienced a thrombotic eventduring or after PTCA. All but two of the 47 c7E3 treated patients had asuccessful PTCA as defined angiographically by a reduction of thelesion(s) to less than 50% luminal diameter narrowing. Of the twounsuccessful dilatations, patient 01-012 had a reduction of a 90%narrowing of the left anterior descending coronary artery to 70%, butfurther dilation was technically not possible. The second patient,(patient 01-019), reviewed below, had an initially successful dilation,but required intracoronary stent placement for a major longitudinaldissection (without evident thrombus). One of the 9 control patients(01-022) experienced thrombotic abrupt closure 15 minutes into theprocedure, requiring emergency coronary artery bypass surgery (CABG),from which he recovered. The other 8 control patients had successfuldilatations to 50% or less residual narrowing.

Patient 01-019 (12-hour infusion group) had a balloon dilatation of a95% lesion of the left circumflex coronary artery with a 50% residualnarrowing. After the procedure, the patient experienced an apparentvasovagal episode, leading to bradycardia, hypotension, and transientasystole. He was returned to the catheterization laboratory and hadurgent intracoronary stent placement for a persistent major longitudinaldissection. The stent became dislodged in the left main coronary artery,and the patient was sent for emergency coronary artery bypass surgery.According to the investigator, there was no evidence of intracoronarythrombosis angiographically or intraoperatively. This patient alsoexperienced a peri-operative myocardial infarction. The patientrecovered and was discharged 8 days after surgery.

There were 3 c7E3 Fab-treated patients who each experienced an isolatedepisode of chest pain post-PTCA of uncertain significance. Patient01-009 (0.25 mg/kg single does group) experienced chest pain 9 hourspost-c7E3, patient 05-003 (12-hour infusion group) experienced angina 21hours post-c7E3, and patient 06-003 (12-hour infusion group) experiencedangina 2 days post-c7E3. The investigators reported that these episodesof chest pain were unrelated to ischemic symptoms signifyingreocclusion.

Patient 02-004 (0.25 mg/kg single does group) experienced prolongedperiods of chest pain prior to c7E3 Fab treatment which continued duringthe PTCA procedure. The following day ECG changes accompanied byelevated cardiac enzymes (drawn the preceding day) indicated that thispatient had experienced a peri-procedural non Q-wave myocardialinfarction (peak creatinine kinase=462, MB fraction=64).

There was one death in the trial which occurred 52 days after c7E3 Fabadministration. Patient 06-002 (6-hour infusion group), who had ahistory of interstitial lung disease, congestive heart failure andunstable angina, underwent successful PTCA of the proximal left anteriordescending coronary artery. During the procedure the patient developedsustained ventricular fibrillation, twice requiring electricaldefibrillation, but thereafter the procedure proceeded uneventfully.After leaving the catheterization laboratory, the patient developedcyanosis, which initially responded to diuretics and oxygen therapy.However, this patient subsequently developed progressive respiratoryimpairment and later required ventilatory support. The patient'ssubsequent hospital course was complicated by sepsis, adult respiratorydistress syndrome, anemia (requiring multiple transfusions), and cardiacischemia. This patient died 52 days post-c7E3 Fab due to multi-systemfailure.

Safety: Stage I and Stage II Observations

FIG. 8 shows the absolute change in hematocrit from baseline to 24 hoursfollowing the end of infusion for all patients by dose group. Forreference, a line indicating the zero change point is shown. Thehematocrit data from one control patient (01-022) and one c7E3Fab-treated patient (01-019) are not plotted because both patientsrequired blood transfusions following urgent coronary bypass surgery inthe first 24 hours (see below). A second lower line at -12 indicates thechange in hematocrit needed to be designated as a minor bleed using theThrombolysis in Myocardial Infarction (TIMI) criteria (Rao et al., J.Am. Coll. Cardiol. 11: 1-11 (1988)). The changes in hematocrit aresimilar between the control patients and all c7E3 Fab dose groups. Table10 summarizes the median change in platelet count at 24 hours followingthe end of infusion. The change in platelet count in the untreatedcontrol and c7E3 Fab-treated groups had similar distributions, with noapparent dose-related effect.

                  TABLE 10    ______________________________________    MEDIAN % CHANGE IN PLATELET COUNT AT 24 HOURS    FOLLOWING THE END OF INFUSION    Dose            % Change Range    ______________________________________    Controls (n = 9)                    -0.7     (-20.2, +13.2)    0.15 mg/kg (n = 5)                    -17.4    (-24.5, +19.5)    0.20 mg/kg (n = 5)                    -11.6    (-20.8, +4.7)    0.25 mg/kg (n = 5)                    +2.9     (-8.9, +4.4)    6 hours* (n = 11)                    -7.7     (-28.3, +19.4)    12 hours* (n = 11)                    0.0      (-24.3, +24.4)    24 hours* (n = 10)                    -3.9     (-9.0, +50.0)    ______________________________________     *Period of infusion (10 μg/min) of c7E3 Fab following a bolus injectio     of 0.25 mg/kg

Discussion of Stage I and Stage II Results

Stage I of this study established that c7E3 exhibits the same doseresponse characteristic in the PTCA population treated with aspirin andheparin as was seen in stable angina patients in a dose escalation trial(Example 3). Chimeric 7E3 produces a dose-dependent blockade of plateletGPIIb/IIIa receptors, and this receptor blockade correlates withinhibition of platelet function. In addition, Stage II resultsdemonstrate that prolonged inhibition of platelet Fab function up to 24hours can be achieved by a continuous infusion. In all patients,platelet functional recovery begins by 6 to 12 hours after cessation ofthe infusion, regardless of the duration of infusion.

The clinical outcome of the c7E3-treated patients, using bothangiographic and clinical endpoints, was considerably better thanexpected based on their risk profile. No patient in the c7E3 treatmentgroup experienced a thrombotic event during or after the procedure. Inaddition, all but 2 patients had angiographically successful procedures.All patients enrolled into Stage II and 6 of the 15 patients enrolled inStage I were high risk patients on the basis of clinical or angiographiccharacteristics. An individual clinical factor (such as unstable angina,diabetes, women over age 65 years) or angiographic lesion-specificcharacteristic (such as type B or C) places a patient at increased riskof complications, and the effect of multiple factors are cumulative. InStage II, 17 treated patients had unstable angina with or withoutadditional clinical or angiographic lesion-specific risk factors. Inaddition, 6 patients in Stage I were identified as having unstableangina. Published series have identified unstable angina patients ashaving a major complication (death, myocardial infarction, urgentcoronary bypass surgery, or repeat PTCA) rate of 10 to 15% (De Feyter,P. J.: Editorial. Am. Heart J. 118: 860-868 (1989) and Rupprecht, H. J.et al. Eur Heart J. 11: 964-973, (1990)). Angiographic characteristicssimilarly are highly predictive of PTCA complications (Ellis, S. G.:Elective coronary angioplasty: technique and complications. In Textbookof Interventional Cardiology, (Ed. E. J. Topol), W. B. Saunders Co.,Philadelphia (1990); De Feyter, P. J. et al., Circulation 83: 927-936(1991); Ellis, S. G. and Topol, E. J, Am. J. Cardiol. 66: 932-937(1990); and ACC/AHA Task Force Report: Guidelines for percutaneoustransluminal coronary angioplasty. J. Am. Coll. Cardiol. 12: 5290545(1988)). Twenty-nine Stage II c7E3-treated patients met the eligibilitycriteria by means of lesion-specific characteristics. Of these, 12patients had one Type B lesion, 14 had 2 or more Type B lesions, andthree had Type C lesions. In addition, many of the patients in the trialhad multiple lesions dilatated in a single or more than one vessel,which also potentially increases the risk of the procedure (Samson, M.et al., Am. Heart J. 120: 1-12 (1990)). On the basis of both the numberand severity of high risk angiographically defined risk factors in thesepatients, ischemic complications would have been expected in the rangeof 10 to 20% (Ellis, S. G.: Elective coronary angioplasty: technique andcomplications. In Textbook of Interventional Cardiology, (Ed. E. J.Topol), W. B. Saunders Co., Philadelphia (1990); De Feyter, P. J.,Circulation 83: 927-936 (1991); Ellis, S. G. and Topol, E. J, Am. J.Cardiol. 66: 932-937 (1990)).

The control group also was comprised of high risk patients. However, ingeneral, the number and severity of risk factors was lower in thecontrol patients. Five of the 9 control patients had single risk factorsof either one type B lesion (4 patients) or unstable angina (1 patient),whereas 26 of 32 Stage II c7E3-treated patients had either a type Clesion or two or more other high risk characteristics. This differencein risk status between the 2 groups is significantly different (Fisher'sexact test p=0.018). Interestingly, the control patient with the abruptclosure (patient 01-022, unstable angina with 2 type B characteristics)was one of 3 patients identified as having more than one risk factor(one control patient had unspecified risk characteristics). Thus,whereas one of three control patients at highest risk had a thromboticevent, none of the 26 c7E3 Fab patients in this highest risk categoryhad a thrombotic event.

This study also demonstrates that the potent antiplatelet effects ofc7E3 can be achieved safely in patients already being treated withintravenous heparin and oral aspirin. Bleeding events were comparable inthe control and c7E3-treated patients with no difference in hematocritchanges from baseline between dosing groups. Other adverse events wereinfrequent and typically of mild or moderate severity. There was onedeath in the trial, and this occurred almost 2 months after c7E3 Fabtreatment in a patient with interstitial lung disease and heart failurewho had progressive respiratory failure following PTCA, complicated bysepsis, adult respiratory distress syndrome, and eventually multipleorgan failure.

Finally, none of the twenty patients in whom results were availableexperienced a human anti-chimeric antibody immune response.

In conclusion, chimeric 7E3 Fab potently inhibits platelet functionsafely in patients treated with aspirin and intravenous heparin who areundergoing PTCA. The antiplatelet action can be maintained for a long as24 hours without a significant increase in bleeding risk and withoutimmune system reactivity. Among patients at high risk of thromboticcomplications, no thrombotic events occurred in the group treated withc7E3, suggesting that c7E3 can reduce the risk of thromboticcomplications in this patient population.

Example 5. Treatment of Abrupt Closure During Coronary Angioplasty.

Abrupt coronary arterial closure during coronary angioplasty is themajor determinant of morbidity and mortality in this procedure. Itoccurs in approximately 3%-6% of elective angioplasty cases (Detre, K.M. et al., Circulation 82: 739-750 (1991)), but has been noted to occurin up to 20%-40% of patients who undergo angioplasty for unstable anginapectoris or after acute myocardial infarction (Ellis, S. G. et al.,Circulation 77: 372-279 (1988); DeFeyter, P. J. et al., Circulation 83:927-936 (1991)). The mechanism of abrupt closure is acute thrombosis atthe arterial site where angioplasty has created or extended an area ofendothelial injury. Usually there are disturbed flow patterns due toaltered geometry of the vessel, often from disruption of the plaquematerial, and there is exposure of subendothelial elements includingintimal and often medial dissection. Since initiation of the thrombusrequires adhesion and aggregation of platelets, chimeric 7E3 Fabantibody fragment was used for the treatment of abrupt coronary arterialclosure complicating a coronary angioplasty procedure.

Case Report

The patient is a 45 year old male physician who had been in excellenthealth previously. Beginning one week prior to his angioplastyprocedure, he had begun to experience chest and neck discomfort. Whenthese symptoms persisted and worsened over several days, he sought theadvice of a colleague. An electrocardiogram (EKG) revealed anteriorprecordial T wave inversions. The patient was then hospitalized in thecoronary care unit of a local hospital and placed on intravenousnitroglycerin and heparin, and oral aspirin. Serial Cardiac isoenzymedeterminations over the next 24 hours did not reveal elevation above thenormal range. Serial EKG recordings over the next two days revealedpersistent flattening of the anterior precordial T waves but noevolutionary changes of myocardial infarction. On the second day afterhospitalization the patient was taken to the cardiac catheterizationlaboratory, where left ventriculography revealed overall normal leftventricular function with a very small hypokinetic area in theanterolateral left ventricular wall, and another hypokinetic area in theinferoposterobasilar zone. The left ventricular ejection fraction was72%. Coronary arteriography demonstrated a left-dominant coronary systemwith a small and totally occluded right coronary artery. There was asignificant stenosis in the mid portion of the left anserior descending(LAD) coronary artery. A small and diffusely diseased diagonal branchoriginated just distal to the mid LAD stenosis.

The patient was returned to the coronary care unit and remained onintravenous nitroglycerin and heparin for another 48 hours. He was painfree during this time, cardiac isoenzymes did not rise, and daily EKGsrevealed only the persistent flattening of the anterior precordial Twaves. He was transferred to Hermann hospital (Houston, Tex.) forangioplasty.

Prior to the angioplasty procedure the patient continued to receiveintravenous nitroglycerin and heparin, oral aspirin, and he was startedon an oral calcium channel blocking agent. The partial thromboplastintime (PTT) had remained in the 70-90 seconds range for several days. Atthe start of his angioplasty procedure the activated clotting time (ACT)was 173 seconds. The patient received 5000 units heparin intravenously.The left coronary ostium was engaged with a number 8 French JL 3.5guiding catheter. The LAD coronary artery was visualized in the caudalright anterior oblique and cranial left anterior oblique projections.The LAD was first instrumented with a 0.018 inch Doppler guidewire(Cardiometrics, Inc., Mountain View, Calif.). This guidewire is used byus routinely for flow monitoring in patients at higher risk for abruptclosure. Flow-velocity signals from the LAD proximal and distal to thelesion were recorded. A 2.5 mm coronary balloon catheter (Intrepid,Baxter, Inc., Irvine, Calif.) was advanced over the Doppler guidewirewhile the wire was held stationary in the coronary artery. The balloonwas positioned so that it straddled the LAD lesion. Sequential briefballoon inflations were made to 6 atmospheres pressure. The severity ofthe stenosis was reduced as visualized by angiography as well as byincrease in the flow velocity signal from a peak flow velocity (APV) of12 cm/sec to 33 cm/sec.

During several minutes of observation following these dilations it wasnoted that the flow signal began to diminish. A contrast injectionrevealed renarrowing of the angioplasty site from elastic recoil, plaquedisruption, and formation of thrombus. The balloon was reintroduced tothe site of the lesion and another balloon inflation was performed. Theartery was reexpanded and the flow signal again returned to an APV of 34cm/sec. During several more minutes of monitoring the signal againdeclined. Within 5 minutes the signal was quite low, at average peakvelocity of 3 cm/sec. The patient began to experience chest pain. TheEKG monitor of an anterior precordial lead revealed ST segmentelevation. Angiography revealed that the artery was completely occluded.The activated clotting time obtained just a few minutes before was 344seconds.

Chimeric 7E3 monoclonal antibody Fab fragment specific for the plateletGP IIb/IIIa receptor (c7E3 Fab, γ₁, κ) was administered. The dose was0.25 mg per kilogram given intravenously over 1 minute. Withinapproximately 1 to 2 minutes after administration of c7E3 Fab, thecoronary flow velocity began to increase. An injection of contrastrevealed restoration of coronary patency with Trombolysis In MyocardialInfarction Trial Grade-1 (TIMI 1) flow. Over the subsequent 15 minutescoronary flow continued to increase and stabilized at an APV of 23cm/sec. Several other injections of contrast demonstrated improvedcoronary flow. The patient's chest pain subsided and the ST segmentobserved in the monitor lead returned to baseline.

Fifteen minutes after administration of c7E3 Fab, an angiogram was madeaccording to protocol. This angiogram revealed TIM13 coronary flow. Theflow velocity signal at this time was 20 cm/sec. Continuous monitoringthrough the subsequent 5 minutes revealed no further improvement in thecoronary flow. During that time the video replay of the angiogramconfirmed that there was a small amount of thrombus still visible at theangioplasty site. For this reason it was decided to administerintracoronary urokinase 250,000 units. This thrombolytic agent wasinfused over approximately the next 10 minutes. During that time therewas no further improvement in flow as measured by the Doppler guidewire.After completion of the intracoronary urokinase infusion, at the 33rdminute after administration of c7E3 Fab, another coronary angiogram wasmade. The artery was patent with TIMI 3 flow. Some moderate but definiteresidual stenosis persisted at the lesion site. In addition, it wasobserved that the thrombus had diminished further in size but had notbeen completely dissolved. The decision was made to perform anotherballoon inflation in order to try to reduce the residual stenosis.

The balloon catheter was again advanced over the guidewire to the siteof the lesion. A final balloon inflation to 6 atmospheres for 2 minuteswas then performed. Then, the balloon catheter was withdrawn while thewire remained in place. The flow signal increased to an APV of 29 cm/secand remained stable over several minutes. An angiogram demonstratedadequate reduction in the residual stenosis which had been present. Theguidewire was then withdrawn proximal to the stenosis and another flowvelocity recording was made. The guidewire, balloon catheter and guidingcatheter were withdrawn. This completed the procedure.

The patient was then taken to the coronary care unit. He remained onoral aspirin, nitrates, a calcium channel blocking agent, andintravenous heparin for several days in order to keep the PTT in the70-90 seconds range. Serial EKGs demonstrated resolution of the anteriorprecordial T wave inversions and all subsequent EKGs were normal. Serialcreatine kinase (CK) isoenzyme values were consistently <100 U/L. Theplatelet count prior to the PTCA procedure was 248,000, and subsequentplatelet counts at 2 h, 6 h, 12 h, 24 h, and 48 h after c7E3 F(ab)administration were 304,000, 279,000, 246,000, 185,000 and 220,000,respectively. Platelet aggregation induced by 10 μM ADP was 73% byoptical densitometry prior to the procedure, and subsequent values at 2h, 6 h, 12 h, 24 h, and 48 h were 0%, 13%, 26%, 45%, and 51%,respectively. One week after the angioplasty procedure, the patient hada follow-up catheterization. The LAD coronary artery was found to bewidely patent with TIMI 3 flow. He was discharged home later that sameday.

Discussion of Case Report

In this patient, the combination of 0.25 mg/kg c7E3 Fab intravenously,250,000 U intracoronary urokinase, and repeat dilatation, successfullytreated the acute ischemic coronary syndrome of abrupt closure duringcoronary angioplasty. These results suggest that antiplatelet therapythat inhibits platelet glycoprotein IIb/IIIa receptor binding andplatelet cross-bridging may be efficacious in helping to achieve stablereperfusion of acutely occluded coronary arteries in similar clinicalsettings.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. A fused gene encoding a chimeric immunoglobulin light chaincomprising:a) a first DNA sequence encoding an immunoglobulin lightchain variable region of the antibody produced by the hybridoma havingATCC accession number HB 8832; and b) a second DNA sequence encoding ahuman light chain constant region.
 2. A host cell comprising the fusedgene of claim
 1. 3. An expression vector comprising a fused gene ofclaim
 1. 4. A host cell comprising an expression vector of claim
 3. 5. Afused gene encoding a chimeric immunoglobulin heavy chain comprising:a)a first DNA sequence encoding an immunoglobulin heavy chain variableregion of the antibody produced by the hybridoma having ATCC accessionnumber HB 8832; and b) a second DNA sequence encoding a human heavychain constant region.
 6. A host cell comprising the fused gene of claim5.
 7. An expression vector comprising a fused gene of claim 5 inexpressible form.
 8. A host cell comprising an expression vector ofclaim
 7. 9. A fused gene encoding a chimeric immunoglobulin heavy chainfragment comprising:a) a first DNA sequence encoding an immunoglobulinheavy chain variable region of the antibody produced by the hybridomahaving ATCC accession number HB 8832; and b) a second DNA sequenceencoding a portion of a human heavy chain constant region,wherein saidfirst and second DNA sequences encode the heavy chain of an Fabfragment.
 10. A host cell comprising the fused gene of claim
 9. 11. Anexpression vector comprising a fused gene of claim
 9. 12. A host cellcomprising an expression vector of claim 11.